乙型肝炎病毒S基因129Leu和145Arg变异株的抗原表达与DNA免疫研究  

Antigen expression and DNA immunization of 129Leu and 145Arg variants of hepatitis B virus S gene

作　　者：何建文[1,2] 武力[1,2] 姚忻[1,2] 李河民 闻玉梅 

机构地区：[1]上海医科大学分子病毒学卫生部重点实验室 [2]中国药品生物制品检定所

出　　处：《中华微生物学和免疫学杂志》1999年第3期201-205,共5页Chinese Journal of Microbiology and Immunology

基　　金：自然科学基金

摘　　要：目的研究我国发现的乙肝病毒Ｓ基因突变株（１２９Ｌｅｕ和１４５Ａｒｇ）表达ＨＢｓＡｇ及ＤＮＡ免疫的特性。方法用自乙肝疫苗免疫无保护婴儿血清中克隆的２株变异Ｓ基因（其中“ａ”决定簇突变，分别为ｎｔ５４０Ａ→Ｔ，ａａ１２９Ｇｌｎ→Ｌｅｕ和ｎｔ５８７Ｇ→Ａ，ａａ１４５Ｇｌｙ→Ａｒｇ）片段，置换已知核苷酸序列并可表达及复制的ＨＢＶ１．２个拷贝全基因野毒株重组质粒ｐ３．８Ⅱ的Ｓ基因。将重组质粒转染ＨｅｐＧ２细胞，用Ａｂｂｏｔ试剂和２４种单抗检测转染细胞培养上清中ＨＢｓＡｇ的结合力。用重组质粒ＤＮＡ肌肉免疫小鼠。结果１２９Ｌｅｕ变异株转染细胞上清对ＨＢｓＡｇ酶联免疫反应检测的吸光度（Ａ）值高于ｐ３．８Ⅱ野毒株，而１４５Ａｒｇ变异株的Ａ值低于ｐ３．８Ⅱ；用２４种单抗测定的总趋势为１２９Ｌｅｕ表达的ＨＢｓＡｇ的Ｓ／Ｃ值高于ｐ３．８Ⅱ，而１４５Ａｒｇ的结果则低于ｐ３．８Ⅱ。三者表达的ＨＢｅＡｇ水平相似。用ＤＮＡ免疫经１２９Ｌｅｕ诱生的抗－ＨＢｓ水平低于野毒株ｐ３．８Ⅱ，但略高于１４５Ａｒｇ。结论１２９Ｌｅｕ变异株表达的ＨＢｓＡｇ与ＨＢｓ多抗及单抗的结合力较野毒株明显增高，但１２９Ｌｅｕ变异株ＤＮＡ免疫诱生的抗－ＨＢｓ水平却低于?Objective To study the HBsAg expression and DNA immunogenicity of 129Leu and 145Arg variants of hepatitis B virus (HBV) S gene discovered in China. Methods The complete S genes of HBV from serum samples with the “a” determinant mutation (nt540A→T, aa129Gln→Leu and nt587G→A, aa145Gly→Arg respectively) were used to substitute the wild type S gene of p3.8Ⅱ, which has 1.2 copies of sequenced HBV genome with expression and replication efficiency. The constructs were separately used to transfect HepG2 cells. HBsAg in supernatant was assayed with Abbott EIA kits and the binding efficiency of HBsAg was analyzed by using 24 monoclonal antibodies (McAb) directed to the S antigens. Plasmid DNAs of constructs were separately used to immunize mice. Results Absorbance ( A ) of HBsAg in the supernatant transfected with variant 129Leu was higher than that of p3.8Ⅱ, while the A value of variant 145Arg was lower than that of p3.8Ⅱ. The binding efficiency (S/C value) with 24 McAb of HBsAg expressed by 129Leu was generally higher than that of p3.8Ⅱ, while the result of 145Arg was lower than that of p3.8Ⅱ. HBeAg expression level among these constructs was similar. Compared with the result of p3.8Ⅱ, anti HBs of DNA immunization induced by variant 129Leu was lower than that of the wild type HBV plasmid DNA. Conclusions HBsAg expressed by variant 129Leu showed higher binding efficiency to the majority of monoclonal antibodies than that of the wild type virus, however, mouse anti HBs induced by variant 129Leu was lower than that of p3.8Ⅱ. The low immunogenicity of variant 129Leu may favor persistent infection in hosts.

关 键 词：乙型肝炎病毒 基因变异 DNA免疫 HBsAg 

分 类 号：R373.21[医药卫生—病原生物学]